The overall objective of the proposed research is to develop a novel treatment for Hepatitis C virus (HCV) infection. HCV remains a major global health care problem, as approximately 170 million people are chronically infected worldwide, with 3.2 million in the United States alone. A vaccine against HCV does not exist, and the best current treatment, a combination of interferon- and ribavirin, is effective in only about 50% of those infected with the most prevalent HCV genotype (genotype 1). Development of treatments and vaccines against this virus are challenging because of its broad genetic diversity and due to its propensity to mutate. Indeed, most of the small molecule drugs being developed for HCV are specific for only a single HCV genotype and drug resistance emerges rapidly. Thus, there is a vital need to develop novel therapies for HCV. The successful treatment of HCV will most likely require a combination of multiple drugs to prevent the generation of viral escape mutants, and RNA interference (RNAi) provides a means for achieving this. RNAi is a sequence-specific post-transcriptional mechanism of gene silencing, which utilizes a variety of small RNAs to cleave cognate mRNAs. The use of vectors encoding short hairpin RNAs (shRNAs) has been the most common strategy employed to provide sustained expression of RNAi effectors. However, over-expression and incomplete processing of shRNAs has led to saturation of the endogenous miRNA pathway, resulting in toxicity. The use of endogenous micro-RNAs (miRNAs) as scaffolds to create so- called artificial miRNAs appears to avoid these problems. We propose to exploit the endogenous miRNA-17-92 cluster as a scaffold for the expression of five artificial miRNAs targeting different regions of the HCV genome. Recombinant adeno-associated virus vectors will be used for delivery of the miRNAs and they will be expressed from a tissue-specific promoter to limit expression to the liver, the site of HCV infection. The artificial miRNAs will be evaluated for gene silencing in normal mice using luciferase reporter plasmids and for inhibition of HCV replication in the HCV cell culture model in vitro. In addition, the ability of the miRNAs t inhibit HCV in a human hepatocyte-chimeric mouse model will be evaluated. We predict that this new combination platform will efficiently inhibit HCV replication and will prevent the major problem that exists with mono-therapies; i.e., the generation of drug-resistant escape mutants.